Understanding plant or animal development requires knowing the regulatory circuitry that orchestrates the gene-expression programs needed to differentiate from a stem cell to all the different cell-types and tissues. Small non-coding RNAs, known as microRNAs (miRNAs), have recently been implicated as components of this regulatory circuitry. MiRNAs are tiny RNAs, about 22 nucleotides in length, that are thought to use elements of the RNA-interference pathway to posttranscriptionally down-regulate the expression of protein-coding genes. Many miRNA genes have been found in animals, where they comprise between 0.5 and 1% of the total genes. Some miRNAs have also been found in plants, and in contrast to the animal miRNAs, regulatory targets have been computationally identified for nearly all of these plant miRNAs. The collaborative research plan of this proposal focuses on the plant miRNAs and their predicted targets, with the broad, long-term objective of understanding the roles of miRNAs in regulating gene expression during plant development. The specific aims of the proposed research are: 1) To examine the phenotypic consequences of disrupting miRNA regulation. 2) To correlate the spatial expression of target mRNAs with the localization of miRNA function. 3) To identify additional plant miRNAs and candidate targets. In experiments of aims #1 and #2, transgenic plants with disrupted or ectopic miRNA regulation will be constructed and subjected to molecular (e.g., in situ hybridizations, molecular reporters) and morphometric analyses. These experiments will determine the biological importance of proper miRNA function and identify developmental roles for miRNA targets. They also will elucidate the molecular bases of any developmental abnormalities, specifically investigating the hypothesized role of miRNAs in clearing key regulatory transcripts from daughter-cell lineages. In experiments of aim #3, additional miRNAs and their targets will be identified by cDNA cloning and subsequent computational analyses. Because so little is known about this newly identified class of genes, speculation is rife on the processes in which they might function in humans. In the same way that previous work on RNA-mediated gene silencing in plants has informed the understanding similar processes in animals, identifying plant miRNAs and studying their biological roles will provide important insights and reagents for understanding the roles of miRNAs in humans and how their dysfunction might contribute to disease. [unreadable] [unreadable]